Methods and Compositions for RNAi Mediated Inhibition of Gene Expression in Mammals

ABSTRACT

Methods and compositions are provided for modulating, e.g., reducing, coding sequence expression in mammals. In the subject methods, an effective amount of an RNAi agent, e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA, is administered to a non-embryonic mammal, e.g., via a hydrodynamic administration protocol. Also provided are RNAi agent pharmaceutical preparations for use in the subject methods. The subject methods and compositions find use in a variety of different applications, including academic and therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.60/307,411 filed Jul. 23, 2001 and U.S. Provisional Patent ApplicationSer. No. 60/360,664 filed Feb. 27, 2002; the disclosures of which areherein incorporated by reference.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contract A1004132awarded by the National Institutes of Health. The Government has certainrights in this invention.

INTRODUCTION Field of the Invention

The field of this invention is RNAi.

BACKGROUND OF THE INVENTION

Double-stranded RNA induces potent and specific gene silencing through aprocess referred to as RNA interference (RNAi) or posttranscriptionalgene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex(RISC), a sequence-specific, multicomponent nuclease that destroysmessenger RNAs homologous to the silencing trigger. RISC is known tocontain short RNAs (approximately 22 nucleotides) derived from thedouble-stranded RNA trigger.

RNAi has become the method of choice for loss-of-function investigationsin numerous systems including, C. elegans, Drosophila, fungi, plants,and even mammalian cell lines. To specifically silence a gene in mostmammalian cell lines, small interfering RNAs (siRNA) are used becauselarge dsRNAs (>30 bp) trigger the interferon response and causenonspecific gene silencing.

To date, the Applicants are not aware of any report of successfulapplication of RNAi technology to non-embryonic mammalian organisms.Demonstration that RNAi works in non-embryonic mammalian organisms wouldprovide for a number of important additional applications for RNAitechnology, including both research and therapeutic applications, and istherefore of intense interest.

Relevant Literature

WO 01/68836. See also: Bernstein et al., RNA (2001) 7: 1509-1521;Bernstein et al., Nature (2001) 409:363-366; Billy et al., Proc. Nat'lAcad. Sci USA (2001) 98:14428-33; Caplan et al., Proc. Nat'l Acad. SciUSA (2001) 98:9742-7; Carthew et al., Curr. Opin. Cell Biol (2001) 13:244-8; Elbashir et al., Nature (2001) 411: 494-498; Hammond et al.,Science (2001) 293:1146-50; Hammond et al., Nat. Ref. Genet. (2001)2:110-119; Hammond et al., Nature (2000) 404:293-296; McCaffrrey et al.,Nature (2002): 418-38-39; and McCaffrey et al., Mol. Ther. (2002)5:676-684; Paddison et al., Genes Dev. (2002) 16:948-958; Paddison etal., Proc. Nat'l Acad. Sci USA (2002) 99:1443-48; Sui et al., Proc.Nat'l Acad. Sci USA (2002) 99:5515-20.

U.S. Patents of interest include U.S. Pat. Nos. 5,985,847 and 5,922,687.Also of interest is WO/11092. Additional references of interest include:Acsadi et al., New Biol. (January 1991) 3:71-81; Chang et al., J. Virol.(2001) 75:3469-3473; Hickman et al., Hum.

Gen. Ther. (1994) 5:1477-1483; Liu et al., Gene Ther. (1999)6:1258-1266; Wolff et al., Science (1990) 247: 1465-1468; and Zhang etal., Hum. Gene Ther. (1999) 10:1735-1737: and Zhang et al., Gene Ther.(1999) 7:1344-1349.

SUMMARY OF THE INVENTION

Methods and compositions are provided for modulating, e.g., reducing,coding sequence expression in mammals. In the subject methods, aneffective amount of an RNAi agent, e.g., an interfering ribonucleic acid(such as an siRNA or shRNA) or a transcription template thereof, e.g., aDNA encoding an shRNA, is administered to a non-embryonic mammal, e.g.,via a hydrodynamic administration protocol. Also provided are RNAi agentpharmaceutical preparations for use in the subject methods. The subjectmethods and compositions find use in a variety of differentapplications, including academic and therapeutic applications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides expression constructs employed in the RNAi experimentsdescribed below.

FIGS. 2A to 2D: RNA interference in adult mice. FIG. 2A) Representativeimages of light emitted from mice co-transfected with the luciferaseplasmid pGL3-Control and either no siRNA (left), luciferase siRNA(middle) or unrelated siRNA (right). A pseudocolor image representingintensity of emitted light (red most and blue least intense)superimposed on a grayscale reference image (for orientation) shows thatRNAi functions in adult mammals. Forty μg of annealed 21-mer siRNAs(Dharmacon) were co-injected into the livers of mice with the 2 μg ofpGL3-Control DNA and 800 units of RNasin (Promega) in 1.8 ml of PBS in5-7 seconds. Seventy two hours after the original injection, mice wereanesthetized and given 3 mg of luciferin intraperitoneally 15 min priorto imaging. FIG. 2B) Summary of siRNA data. Mice receiving luciferasesiRNA emitted significantly less light than untreated controls. Aone-way ANOVA analysis with a post hoc Fisher's test was conducted. Theuntreated and unrelated siRNA groups were statistically similar. FIG.2C) pShh1-Ff1 (center) but not pShh1-Ff1rev (right) reduced luciferaseexpression in mice compared to the untreated control (left). 10 μg ofpShh1-Ff1 or pShh1-rev were co-injected with 40 μg of pLuc-NSSB in 1.8ml of PBS. FIG. 2D) Quantitation of pShh1 data. Animals were treatedaccording to NIH Guidelines for Animal Care and the Guidelines ofStanford University.

FIG. 3 provides a schematic representation of the constructs employed inthe morpholino phosporamidate antisense HCV inhibition assay performedin the Experimental Section, below.

FIG. 4 provides background information of the mechanism of antisenseinhibitors.

FIGS. 5A to 5F provide graphical results of a morpholino phosporamidateantisense HCV inhibition assay performed according to the subjectinvention.

DEFINITIONS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a genomic integrated vector, or“integrated vector”, which can become integrated into the chromsomal DNAof the host cell. Another type of vector is an episomal vector, i.e., anucleic acid capable of extra-chromosomal replication in an appropriatehost, e.g., a eukaryotic or prokaryotic host cell. Vectors capable ofdirecting the expression of genes to which they are operatively linkedare referred to herein as “expression vectors”. In the presentspecification, “plasmid” and “vector” are used interchangeably unlessotherwise clear from the context.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as applicable tothe embodiment being described, single-stranded (such as sense orantisense) and double-stranded polynucleotides.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide ofthe present invention, including both exon and (optionally) intronsequences. A “recombinant gene” refers to nucleic acid encoding suchregulatory polypeptides, that may optionally include intron sequencesthat are derived from chromosomal DNA. The term “intron” refers to a DNAsequence present in a given gene that is not translated into protein andis generally found between exons. As used herein, the term“transfection” means the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer.

A “protein coding sequence” or a sequence that “encodes” a particularpolypeptide or peptide, is a nucleic acid sequence that is transcribed(in the case of DNA) and is translated (in the case of mRNA) into apolypeptide in vitro or in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from procaryotic or eukaryoticmRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and evensynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

Likewise, “encodes”, unless evident from its context, will be meant toinclude DNA sequences that encode a polypeptide, as the term istypically used, as well as DNA sequences that are transcribed intoinhibitory antisense molecules.

The term “loss-of-function”, as it refers to genes inhibited by thesubject RNAi method, refers a diminishment in the level of expression ofa gene when compared to the level in the absence of the RNAi agent.

The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein coding sequence results fromtranscription and translation of the coding sequence.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

By “recombinant virus” is meant a virus that has been geneticallyaltered, e.g., by the addition or insertion of a heterologous nucleicacid construct into the particle.

As used herein, the terms “transduction” and “transfection” are artrecognized and mean the introduction of a nucleic acid, e.g., anexpression vector, into a recipient cell by nucleic acid-mediated genetransfer. “Transformation”, as used herein, refers to a process in whicha cell's genotype is changed as a result of the cellular uptake ofexogenous DNA or RNA, and, for example, the transformed cell expresses adsRNA construct.

“Transient transfection” refers to cases where exogenous DNA does notintegrate into the genome of a transfected cell, e.g., where episomalDNA is transcribed into mRNA and translated into protein.

A cell has been “stably transfected” with a nucleic acid construct whenthe nucleic acid construct is capable of being inherited by daughtercells.

As used herein, a “reporter gene construct” is a nucleic acid thatincludes a “reporter gene” operatively linked to at least onetranscriptional regulatory sequence. Transcription of the reporter geneis controlled by these sequences to which they are linked. The activityof at least one or more of these control sequences can be directly orindirectly regulated by the target receptor protein. Exemplarytranscriptional control sequences are promoter sequences. A reportergene is meant to include a promoter-reporter gene construct that isheterologously expressed in a cell.

As used herein, “transformed cells” refers to cells that havespontaneously converted to a state of unrestrained growth, i.e., theyhave acquired the ability to grow through an indefinite number ofdivisions in culture. Transformed cells may be characterized by suchterms as neoplastic, anaplastic and/or hyperplastic, with respect totheir loss of growth control. For purposes of this invention, the terms“transformed phenotype of malignant mammalian cells” and “transformedphenotype ” are intended to encompass, but not be limited to, any of thefollowing phenotypic traits associated with cellular transformation ofmammalian cells: immortalization, morphological or growthtransformation, and tumorigenicity, as detected by prolonged growth incell culture, growth in semi-solid media, or tumorigenic growth inimmuno-incompetent or syngeneic animals.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

As used herein, “immortalized cells” refers to cells that have beenaltered via chemical, genetic, and/or recombinant means such that thecells have the ability to grow through an indefinite number of divisionsin culture.

The “growth state” of a cell refers to the rate of proliferation of thecell and the state of differentiation of the cell.

“Inhibition of gene expression” refers to the absence (or observabledecrease) in the level of protein and/or mRNA product from a targetgene. “Specificity” refers to the ability to inhibit the target genewithout manifest effects on other genes of the cell. The consequences ofinhibition can be confirmed by examination of the outward properties ofthe cell or organism (as presented below in the examples) or bybiochemical techniques such as RNA solution hybridization, nucleaseprotection, Northern hybridization, reverse transcription, geneexpression monitoring with a microarray, antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blotting, radiolmmunoassay (RIA),other immunoassays, and fluorescence activated cell analysis (FACS). ForRNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.

Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of administered activeagent and longer times after administration of active agent may resultin inhibition in a smaller fraction of cells (e.g., at least 10%, 20%,50%, 75%, 90%, or 95% of targeted cells). Quantitation of geneexpression in a cell may show similar amounts of inhibition at the levelof accumulation of target mRNA or translation of target protein. As anexample, the efficiency of inhibition may be determined by assessing theamount of gene product in the cell: mRNA may be detected with ahybridization probe having a nucleotide sequence outside the region usedfor the inhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods and compositions are provided for modulating, e.g., reducing,coding sequence expression in mammals. In the subject methods, aneffective amount of an RNAi agent, e.g., an interfering ribonucleic acid(such as an siRNA or shRNA) or a transcription template thereof, e.g., aDNA encoding an shRNA, is administered to a non-embryonic mammal, e.g.,via a hydrodynamic administration protocol. Also provided are RNAi agentpharmaceutical preparations for use in the subject methods. The subjectmethods and compositions find use in a variety of differentapplications, including academic and therapeutic applications.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, representativemethods, devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the components that aredescribed in the publications which might be used in connection with thepresently described invention.

RNAi in Non-Embryonic Mammals

As summarized above, the subject invention provides methods ofperforming RNAi in non-embryonic mammals. In further describing thisaspect of the subject invention, the subject methods of RNAi innon-embryonic mammals are described first in greater detail, followed bya review of various representative applications in which the subjectinvention finds use as well as kits that find use in practicing thesubject invention.

Methods

As indicated above, one aspect of the subject invention provides methodsof employing RNAi to modulate expression of a target gene or genes in anon-embryonic mammalian host. In many embodiments, the subject inventionprovides methods of reducing expression of one or more target genes in anon-embryonic mammalian host organism. By reducing expression is meantthat the level of expression of a target gene or coding sequence isreduced or inhibited by at least about 2-fold, usually by at least about5-fold, e.g., 10-fold, 15-fold, 20-fold, 50-fold, 100-fold or more, ascompared to a control. In certain embodiments, the expression of thetarget gene is reduced to such an extent that expression of the targetgene/coding sequence is effectively inhibited. By modulating expressionof a target gene is meant altering, e.g., reducing,transcription/translation of a coding sequence, e.g., genomic DNA, mRNAetc., into a polypeptide, e.g., protein, product.

The subject invention provides methods of modulating expression of atarget gene in a non-embryonic mammalian organism. By non-embryonicmammalian organism is meant a mammalian organism or host that is not anembryo, i.e., is at a stage of development that is later in time thanthe embryonic stage of development. As such, the host organism may be afetus, but is generally a host organism in a post natal stage ofdevelopment, e.g., juvenile, adult, etc.

In practicing the subject methods, an effective amount of an RNAi agentis administered to the host organism to modulate expression of a targetgene in a desirable manner, e.g., to achieve the desired reduction intarget cell gene expression.

By RNAi agent is meant an agent that modulates expression of a targetgene by a RNA interference mechanism. The RNAi agents employed in oneembodiment of the subject invention are small ribonucleic acid molecules(also referred to herein as interfering ribonucleic acids), i.e.,oligoribonucleotides, that are present in duplex structures, e.g., twodistinct oligoribonucleotides hybridized to each other or a singleribooligonucleotide that assumes a small hairpin formation to produce aduplex structure. By oligoribonucleotide is meant a ribonucleic acidthat does not exceed about 100 nt in length, and typically does notexceed about 75 nt length, where the length in certain embodiments isless than about 70 nt. Where the RNA agent is a duplex structure of twodistinct ribonucleic acids hybridized to each other, e.g., an siRNA(such as d-siRNA as described in copending application Ser. No.60/377,704; the disclosure of which is herein incorporated byreference), the length of the duplex structure typically ranges fromabout 15 to 30 bp, usually from about 15 to 29 bp, where lengths betweenabout 20 and 29 bps, e.g., 21 bp, 22 bp, are of particular interest incertain embodiments. Where the RNA agent is a duplex structure of asingle ribonucleic acid that is present in a hairpin formation, i.e., ashRNA, the length of the hybridized portion of the hairpin is typicallythe same as that provided above for the siRNA type of agent or longer by4-8 nucleotides. The weight of the RNAi agents of this embodimenttypically ranges from about 5,000 daltons to about 35,000 daltons, andin many embodiments is at least about 10,000 daltons and less than about27,500 daltons, often less than about 25,000 daltons.

In certain embodiments, instead of the RNAi agent being an interferingribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAiagent may encode an interfering ribonucleic acid, e.g., an shRNA, asdescribed above. In other words, the RNAi agent may be a transcriptionaltemplate of the interfering ribonucleic acid. In these embodiments, thetranscriptional template is typically a DNA that encodes the interferingribonucleic acid. The DNA may be present in a vector, where a variety ofdifferent vectors are known in the art, e.g., a plasmid vector, a viralvector, etc.

The RNAi agent can be administered to the non-embryonic mammalian hostusing any convenient protocol, where the protocol employed is typicallya nucleic acid administration protocol, where a number of different suchprotocols are known in the art. The following discussion provides areview of representative nucleic acid administration protocols that maybe employed. The nucleic acids may be introduced into tissues or hostcells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intra-muscular administration, as described by Furth et al. (1992),Anal Biochem 205:365-368. The nucleic acids may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992), Nature 356:152-154), where gold microprojectiles arecoated with the DNA, then bombarded into skin cells. Expression vectorsmay be used to introduce the nucleic acids into a cell. Such vectorsgenerally have convenient restriction sites located near the promotersequence to provide for the insertion of nucleic acid sequences.Transcription cassettes may be prepared comprising a transcriptioninitiation region, the target gene or fragment thereof, and atranscriptional termination region. The transcription cassettes may beintroduced into a variety of vectors, e.g. plasmid; retrovirus, e.g.lentivirus; adenovirus; and the like, where the vectors are able totransiently or stably be maintained in the cells, usually for a periodof at least about one day, more usually for a period of at least aboutseveral days to several weeks.

For example, the RNAi agent can be fed directly to, injected into, thehost organism containing the target gene. The agent may be directlyintroduced into the cell (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, etc. Methods for oral introductioninclude direct mixing of RNA with food of the organism. Physical methodsof introducing nucleic acids include injection directly into the cell orextracellular injection into the organism of an RNA solution. The agentmay be introduced in an amount which allows delivery of at least onecopy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of the agent may yield more effective inhibition; lowerdoses may also be useful for specific applications.

In certain embodiments, a hydrodynamic nucleic acid administrationprotocol is employed. Where the agent is a ribonucleic acid, thehydrodynamic ribonucleic acid administration protocol described indetail below is of particular interest. Where the agent is adeoxyribonucleic acid, the hydrodynamic deoxyribonucleic acidadministration protocols described in Chang et al., J. Virol. (2001)75:3469-3473; Liu et al., Gene Ther. (1999) 6:1258-1266; Wolff et al.,Science (1990) 247: 1465-1468; Zhang et al., Hum. Gene Ther. (1999)10:1735-1737: and Zhang et al., Gene Ther. (1999) 7:1344-1349; are ofinterest.

Additional nucleic acid delivery protocols of interest include, but arenot limited to: those described in U.S. Patents of interest include U.S.Pat. Nos. 5,985,847 and 5,922,687 (the disclosures of which are hereinincorporated by reference); WO/11092;. Acsadi et al., New Biol. (1991)3:71-81; Hickman et al., Hum. Gen. Ther. (1994) 5:1477-1483; and Wolffet al., Science (1990) 247: 1465-1468; etc.

Depending n the nature of the RNAi agent, the active agent(s) may beadministered to the host using any convenient means capable of resultingin the desired modulation of target gene expression. Thus, the agent canbe incorporated into a variety of formulations for therapeuticadministration. More particularly, the agents of the present inventioncan be formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents, and maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols. As such,administration of the agents can be achieved in various ways, includingoral, buccal, rectal, parenteral, intraperitoneal, intradermal,transdermal, intracheal, etc., administration.

In pharmaceutical dosage forms, the agents may be administered alone orin appropriate association, as well as in combination, with otherpharmaceutically active compounds. The following methods and excipientsare merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Preferred dosages for a given compound arereadily determinable by those of skill in the art by a variety of means.

Administration of an effective amount of an RNAi agent to anon-embryonic mammalian host according as described above results in amodulation of target gene(s) expression, e.g., a reduction of targetgene(s) expression, as described above.

The above described methods work in any mammal, where representativemammals of interest include, but are not limited to: ungulates or hoovedanimals, e.g., cattle, goats, pigs, sheep, etc.; rodents, e.g.,hamsters, mice, rats, etc.; lagomorphs, e.g., rabbits; primates, e.g.,monkeys, baboons, humans, etc.; and the like.

The above described methods find use in a variety of differentapplications, representative types of which are now described in greaterdetail below.

Utility

The subject methods find use in a variety of different applications,where representative applications include both academic/researchapplications and therapeutic applications. Each of these types ofrepresentative applications is described more fully below.

Academic/Research Applications

The subject methods find use in a variety of different types ofacademic, research applications, in which one desires to modulateexpression of one or more target genes (coding sequences) in a mammalianhost, e.g., to determine the function of a target gene/coding sequencein a mammalian host. The subject methods find particular use in“loss-of-function” type assays, where one employs the subject methods toreduce or decrease or inhibit expression of one or more targetgenes/coding sequences in a mammalian host.

As such, one representative utility of the present invention is as amethod of identifying gene function in a non-embryonic mammal, where anRNAi agent is administered to a mammal according to the presentinvention in order to inhibit the activity of a target gene ofpreviously unknown function. Instead of the time consuming and laboriousisolation of mutants by traditional genetic screening, functionalgenomics using the subject methods determines the function ofuncharacterized genes by administering an RNAi agent to reduce theamount and/or alter the timing of target gene activity. Such methods canbe used in determining potential targets for pharmaceutics,understanding normal and pathological events associated withdevelopment, determining signaling pathways responsible for postnataldevelopment/aging, and the like. The increasing speed of acquiringnucleotide sequence information from genomic and expressed gene sources,including total sequences for mammalian genomes, can be coupled with useof the subject methods to determine gene function in a live mammalianorganism. The preference of different organisms to use particularcodons, searching sequence databases for related gene products,correlating the linkage map of genetic traits with the physical map fromwhich the nucleotide sequences are derived, and artificial intelligencemethods may be used to define putative open reading frames from thenucleotide sequences acquired in such sequencing projects.

A simple representative assay inhibits gene expression according to thepartial sequence available from an expressed sequence tag (EST).Functional alterations in growth, development, metabolism, diseaseresistance, or other biological processes would be indicative of thenormal role of the EST's gene product. The function of the target genecan be assayed from the effects it has on the mammal when gene activityis inhibited.

If a characteristic of an organism is determined to be geneticallylinked to a polymorphism through RFLP or QTL analysis, the presentinvention can be used to gain insight regarding whether that geneticpolymorphism might be directly responsible for the characteristic. Forexample, a fragment defining the genetic polymorphism or sequences inthe vicinity of such a genetic polymorphism can be employed to producean RNAi agent, which agent can then be administered to the mammal, andwhether an alteration in the characteristic is correlated withinhibition can be determined.

The present invention is useful in allowing the inhibition of essentialgenes. Such genes may be required for organism viability at onlyparticular stages of development or cellular compartments. Thefunctional equivalent of conditional mutations may be produced byinhibiting activity of the target gene when or where it is not requiredfor viability. The invention allows addition of an RNAi agent atspecific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

In situations where alternative splicing produces a family oftranscripts that are distinguished by usage of characteristic exons, thepresent invention can target inhibition through the appropriate exons tospecifically inhibit or to distinguish among the functions of familymembers. For example, a hormone that contained an alternatively splicedtransmembrane domain may be expressed in both membrane bound andsecreted forms. Instead of isolating a nonsense mutation that terminatestranslation before the transmembrane domain, the functional consequencesof having only secreted hormone can be determined according to theinvention by targeting the exon containing the transmembrane domain andthereby inhibiting expression of membrane-bound hormone.

Therapeutic Applications

The subject methods also find use in a variety of therapeuticapplications in which it is desired to modulate, e.g., one or moretarget genes in a whole mammal or portion thereof, e.g., tissue, organ,etc. In such methods, an effective amount of an RNAi active agent isadministered to the host mammal. By effective amount is meant a dosagesufficient to modulate expression of the target gene(s), as desired. Asindicated above, in many embodiments of this type of application, thesubject methods are employed to reduce/inhibit expression of one or moretarget genes in the host in order to achieve a desired therapeuticoutcome.

Depending on the nature of the condition being treated, the target genemay be a gene derived from the cell, an endogenous gene, apathologically mutated gene, e.g. a cancer causing gene, a transgene, ora gene of a pathogen which is present in the cell after infectionthereof. Depending on the particular target gene and the dose of RNAiagent delivered, the procedure may provide partial or complete loss offunction for the target gene.

Lower doses of injected material and longer times after administrationof RNAi agent may result in inhibition in a smaller fraction of cells.

The subject methods find use in the treatment of a variety of differentconditions in which the modulation of target gene expression in amammalian host is desired. By treatment is meant that at least anamelioration of the symptoms associated with the condition afflictingthe host is achieved, where amelioration is used in a broad sense torefer to at least a reduction in the magnitude of a parameter, e.g.symptom, associated with the condition being treated. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g. preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the condition, or at least the symptoms thatcharacterize the condition.

A variety of hosts are treatable according to the subject methods.Generally such hosts are “mammals” or “mammalian,” where these terms areused broadly to is describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In many embodiments, the hosts will behumans.

The present invention is not limited to modulation of expression of anyspecific type of target gene or nucleotide sequence. Representativeclasses of target genes of interest include but are not limited to:developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors,cytokines/lymphokines and their receptors, growth/differentiationfactors and their receptors, neurotransmitters and their receptors);oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB,EBRB2, ETSI, ETS1, ETV6, FOR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN,MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, TALI,TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA 1, BRCA2, MADH4,MCC, NF 1, NF2, RB 1, TP53, and WTI); and enzymes (e.g., ACC synthasesand oxidases, ACP desaturases and hydroxylases, ADP-glucosepyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, chalcone synthases,chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNApolymerases, galactosidases, glucanases, glucose oxidases, granule-boundstarch synthases, GTPases, helicases, hemicellulases, integrases,inulinases, invertases, isomerases, kinases, lactases, Upases,lipoxygenases, lyso/ymes, nopaline synthases, octopine synthases,pectinesterases, peroxidases, phosphatases, phospholipases,phosphorylases, phytases, plant growth regulator synthases,polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOs, topoisomerases, andxylanases); chemokines (e.g. CXCR4, CCR5), the RNA component oftelomerase, vascular endothelial growth factor (VEGF), VEGF receptor,tumor necrosis factors nuclear factor kappa B, transcription factors,cell adhesion molecules, Insulin-like growth factor, transforming growthfactor beta family members, cell surface receptors, RNA binding proteins(e.g. small nucleolar RNAs, RNA transport factors), translation factors,telomerase reverse transcriptase); etc.

Kits

Also provided are reagents and kits thereof for practicing one or moreof the above-described methods. The subject reagents and kits thereofmay vary greatly. Typically, the kits at least include an RNAi agent asdescribed above.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

Hydrodynamic Administration of Naked RNA

Also provided by the subject invention are methods and compositions forthe in vivo introduction of a naked nucleic acid, e.g. ribonucleic acid,deoxyribonucleic or chemically modified nucleic acids (including, butnot limited to, morpholino, peptide nucleic acids, methylphosphonate,phosphorothioate or 2′-Omethyl oligonucleotides), into the target cellof a vascularized organism, e.g. a mammal. These methods of the subjectinvention are conveniently referred to as “hydrodynamic” methods.

In one embodiment of the subject methods, an aqueous formulation of anaked nucleic acid and an RNase inhibitor is administered into thevascular system of the organism. In many embodiments, the aqueousformulation also includes a competitor ribonucleic acid, e.g. anon-capped non-polyadenylated ribonucleic acid. In yet otherembodiments, codelivery of DNA capable of being transcribed into the RNAmolecule with candidate modulatory agents is performed without an RNaseinhibitor or competitor ribonucleic acid, where the modulatory agent andthe DNA may or may not be delivered as a single composition. The subjectmethods find use in a variety of different applications, including bothresearch and therapeutic applications, and are particularly suited foruse in the in vivo delivery of a ribonucleic acid into a hepatic cell,e.g. for liver targeted in vivo delivery of nucleic acids.

In further describing this aspect of the subject invention, the subjectmethods will be described first followed by a description ofrepresentative applications in which the subject methods find use andkits for use in practicing the subject methods.

Methods

As summarized above, the subject invention provides a method for the invivo introduction of a nucleic acid, e.g. a ribonucleic acid, into atarget cell present in a vascularized multi-cellular organism. By invivo introduction is meant that, in the subject methods, the target cellinto which the nucleic acid is introduced is one that is present in themulti-cellular organism, i.e., it is not a cell that is separated from,e.g. removed from, the multi-cellular organism. As such, the subjectmethods are distinct from in vitro nucleic acid transfer protocols, inwhich a nucleic acid is introduced into a cell or cells separated fromthe multi-cellular organism from which they originated, e.g. are inculture. In other words, the subject methods are not methods of in vitronucleic acid transfer.

By introduction of the nucleic acid is meant that the nucleic acid,e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or anon-naturally occurring nucleic acid analog, is inserted into thecytoplasm of the target cell. In other words, the nucleic acid is movedfrom the outside of the target cell to the inside of the target cellacross the cell membrane.

By vascularized multi-cellular organism is meant a multi-cellularorganism that includes a vascular system. Multi-cellular organisms ofinterest include plants and animals, where animals are of particularinterest, particularly vertebrate animals that have a vascular systemmade up of a system of veins and arteries through which blood is flowed,e.g. in response to the beating of a heart. Animals of interest aremammals in many embodiments. Mammals of interest include; rodents, e.g.mice, rats; livestock, e.g. pigs, horses, cows, etc., pets, e.g. dogs,cats; and primates, e.g. humans. In certain embodiments, themulti-cellular organism is a human. In other embodiments, themulti-cellular organism is a non-human mammal, e.g. a rodent, such as amouse, rat, etc.

As mentioned above, the subject methods are, in the broadest sense,suitable for introduction of nucleic acids into the target cell of ahost. The term “nucleic acid” as used herein means a polymer composed ofnucleotides, e.g. deoxyribonucleotides or ribonucleotides, or compoundsproduced synthetically (e.g. PNA as described in U.S. Pat. No. 5,948,902and the references cited therein) which can hybridize with naturallyoccurring nucleic acids in a sequence specific manner analogous to thatof two naturally occurring nucleic acids. The terms “ribonucleic acid”and “RNA” as used herein mean a polymer composed of ribonucleotides. Theterms “deoxyribonucleic acid” and “DNA” as used herein mean a polymercomposed of deoxyribonucleotides.

The subject methods are particularly suited for use in the delivery of aribonucleic acid into a target cell of a multi-cellular organism. Assuch, the methods will now be further described in terms of the deliveryof ribonucleic acids. However, the following protocols are also suitablefor use in the delivery of other nucleic acids, e.g. DNAs (such asplasmid DNA), etc.

In practicing the subject methods, an aqueous composition of theribonucleic acid in which the ribonucleic acid is present as a nakedribonucleic acid is administered to the vascular system of themulti-cellular organism or host. In many embodiments, the naked RNAaqueous composition or formulation is administered to the vein of thehost, i.e. the naked RNA formulation is intravenously administered. Incertain embodiments, the naked RNA formulation is intravenouslyadministered to the host via high pressure injection. By high pressureinjection is meant that the aqueous formulation is intravenouslyintroduced at an elevated pressure, where the elevated pressure isgenerally at least about 20, usually at least about 30 mmHg. In manyembodiments, the elevated pressure ranges from about 10 to 50 mm Hg,where 40 to 50 mm Hg is often preferred. Methods of administeringaqueous formulations under high pressure, such as those described above,are described in the references listed in the relevant literaturesection, supra.

As mentioned above, the RNA or DNA that is to be introduced into thetarget cell via the subject methods is present in the aqueousformulation as naked RNA. By “naked” is meant that the RNA is free fromany delivery vehicle that can act to facilitate entry into the targetcell. For example, the naked RNAs or DNAs delivered in the subjectmethods are free from any material that promotes transfection, such asliposomal formulations, charged lipids or precipitating agents, e.g.they are not complexed to colloidal materials (including liposomalpreparations). In addition, the naked RNAs of the subject invention arenot contained in a vector that would cause integration of the RNA intothe target cell genome, i.e. they are free of viral sequences orparticles that carry genetic information.

The naked RNAs that may be delivered via the subject invention may varywidely in length, depending on their intended purpose, e.g. the proteinthey encode, etc. Generally, the naked RNAs will be at least about 10 ntlong, usually at least about 30 nt long and more usually at least about35 nt long, where the naked RNAs may be as long as 20,000 nt or longer,but generally will not exceed about 10,000 nt long and usually will notexceed about 6,000 nt long. In certain embodiments where the naked RNAis an RNAi agent, as described above, the length of the RNA ranges fromabout 10 to 50 nt, often from about 10 to 40 nt, and more often fromabout 15 to 30 nt, including 15 to 25 nt, such as 20 to 25 nt, e.g., 21or 22 nt.

The naked RNAs that may be introduced into a target cell according tothe subject methods may or may not encode a protein, i.e. may or may notbe capable of being translated into a protein upon introduction into thetarget cell. In those embodiments where the naked RNA is capable ofbeing translated into a protein following introduction into the targetcell, the naked RNA may or may not be capped, it may include an IRESdomain, etc. However, in many particular protocols of this embodiment,the naked RNA is capped. Furthermore, the RNA in these embodimentsgenerally includes at least a polyadenylation signal, and in manyembodiments is polyadenylated, where the polyA tail, when present,generally ranges in length from about 10 to 300, usually from about 30to 50. Further description of the naked RNAs is provided infra.

As mentioned above, an aqueous formulation of the naked RNA isintravascularly, usually intravenously, administered to the host. In theaqueous formulations employed in the subject methods, an effectiveamount of the naked RNA is combined with an aqueous delivery vehicle. Byeffective amount is meant an amount that is sufficient to provide forthe desired amount of transfer into the target cell, e.g. to provide thedesired outcome, such as desired amount of protein expression. In manyembodiments, the amount of naked RNA present in the aqueous formulationis at least about 5 micrograms, usually at least about 10 micrograms andmore usually at least about 20 micrograms, where the amount may be asgreat as 10 milligrams or greater, but generally does not exceed about 1milligram and usually does not exceed about 200 micrograms.

Aqueous delivery vehicles of interest include: water, saline andbuffered media. Specific vehicles of interest include: sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, phosphate buffered saline, etc. The aqueous delivery vehiclesmay further include preservatives and other additives, e.g.antimicrobials, antioxidants, chelating agents, inert gases, nutrientreplenishers, electrolyte replenishers, divalent cations, such asmagnesium, calcium and manganese, etc. Of particular interest in manyembodiments is the use of buffered salt solutions arepseudophysiological.

A feature of certain embodiments of the subject methods is that thenaked RNA is introduced into the vascular system of the multi-cellularorganism in combination with an RNase inhibitor. By RNase inhibitor ismeant a compound or agent that at least reduces the activity of, if notcompletely inactivates, an RNase activity in the multi-cellularorganism. In many embodiments, the RNase inhibitor is a proteininhibitor of RNase, where the human placental RNase inhibitor is ofparticular interest. The protein RNase inhibitor may be purified from anatural source or synthetically produced, e.g. via recombinanttechniques. Human placental RNase inhibitor may be obtained from avariety of different sources under a variety of different tradenames,where representative sources include: Promega, Inc., Strategene, Inc.,Fisher Scientific, Inc., and the like.

While the RNase inhibitor may, in certain embodiments, be administeredto the host in a composition separate from the aqueous naked RNAcomposition, in many embodiments the RNase inhibitor is present in theaqueous naked RNA composition. The amount of RNase inhibitor that ispresent in the aqueous composition is sufficient to provide for thedesired uptake of the naked RNA. Where the RNase inhibitor is a proteininhibitor, the concentration of the inhibitor in the aqueous compositionthat is introduced into the multi-cellular organism during practice ofthe subject methods may range from about 4 to 4,000 units, usually fromabout 400 to 4,000 units and more usually from about 400 to 1,500 units.

In certain embodiments, the naked RNA and RNase inhibitor areadministered in conjunction with a competitor RNA. By competitor RNA ismeant an RNA that is capable of serving as a competitive inhibitor ofRNase activity. In many embodiments, the competitor RNA is uncapped andnon-polyadenylated. By uncapped is meant that the competitor RNA lacksthe cap structure found at the 5′ end of eukaryotic messenger RNA, i.e.it lacks a 5′ 7 methyl G. By non-polyadenylated is meant that thecompetitor RNA lacks a polyA tail or domain of polyadenylation at its 3′end, as is found in eukaryotic messenger RNA. The length of thecompetitor RNA may vary, but is generally at least about 70 nt, usuallyat least about 200 nt and more usually at least about 1,500 nt, wherethe length may be as great as 10,000 nt or greater, but generally doesnot exceed about 3,500 nt and usually does not exceed about 1,500 nt.The concentration of competitor RNA in the aqueous composition issufficient to provide for the desired protection of the naked RNA (e.g.via competition for binding by RNase), and in many embodiments rangesfrom about 10 μg/ml to 10 mg/ml, usually from about 20 to 200 μg/ml andmore usually from about 40 to 150 μg/ml.

The subject methods result in highly efficient transfer of theadministered RNA into the cytoplasm of the target cell(s). The subjectmethods are particularly suited for transferring RNA into the cytoplasmof liver or hepatic cells and non-parenchymal cells in the liver. Assuch, in many embodiments the subject methods are in vivo methods ofachieving high level nucleic acid, e.g. RNA, transfer into hepatic cellsor liver tissue.

The nucleic acid that is introduced into the target cell via the subjectmethods is short lived once inside the target cell. Depending on theparticular nature of the nucleic acid, the half life the nucleic acidfollowing introduction via the subject methods generally ranges fromabout 30 sec to 10 days, usually from about 1 min to 24 hrs and moreusually from about 5 min to 10 hrs. As such, where the nucleic acid isan RNA encoding a protein of interest, protein expression followingintroduction via the subject method is transient, typically lasting fora period of time ranging from about 1 min to 3 days, usually from about5 min to 24 hrs. As such, in many embodiments of the subject methods,the subject methods are methods of providing for transient proteinexpression from a transgene, where protein expression is equal to RNAlifetime. Nonetheless, the protein expressed may have a longer lifetime,depending on the nature of the particular protein.

Utility

The subject methods find use in a variety of different applications inwhich the efficient in vivo transfer of a naked nucleic acid into atarget cell is desired. Applications in which the subject methods finduse include both therapeutic and research applications. Therapeuticapplications of interest include gene therapy applications, vaccinationapplications, and the like. Research applications of interest includethe production of animal models for particular conditions, e.g. RNAviral infections, the observation of gene expression on phenotypes toelucidate gene function, etc. Other applications in which the subjectinvention finds use include the development of antisense, ribozyme andchimeraplasty (i.e. the repair of genes via RNA/DNA chimeras (see e.g.Yoon et al., Proc Natl Acad Sci U S A (1996) 93(5):2071-6; Cole-Strausset al., Science (1996) 273(5280):1386-9; and Zhu et al., Proc Natl AcadSci U S A (1999) 96(15):8768-73) therapeutics, as well as interferingRNA (RNA whose presence in the cell prevents the translation of similarRNAs, (See e.g. Wianny et al., Nat Cell Biol (2000) 2(2):70-5; and SiQunet al., Nature (1998) 391: 806 - 811) therapeutics.

One type of application in which the subject methods find use is in thesynthesis of polypeptides, e.g. proteins, of interest from a targetcell, particularly the transient expression of a polypeptide. In suchapplications, a nucleic acid that encodes the polypeptide of interest incombination with requisite and/or desired expression components, e.g. 5′cap structures, IRES domains, polyA signals or tails, etc., isintroduced into the target cell via in vivo administration to themulti-cellular organism in which the target cell resides, where thetarget cell is to serve as an expression host for expression of thepolypeptide. For example, where the naked nucleic acid administered bythe subject methods is RNA, the RNA is an RNA that is capable of beingtranslated in the cytoplasm of the target cell into the protein encodedby the sequence contained in the RNA. The RNA may be capped or uncapped,where when it is uncapped it generally includes an IRES sequence. TheRNA also generally further includes a polyA tail, where the length ofthe polyA tail typically ranges from about 10 to 300, usually from about30 to 50 nt. Following in vivo administration and subsequentintroduction into the target cell, the multi-cellular organism, andtargeted host cell present therein, is then maintained under conditionssufficient for expression of the protein encoded by the transferred RNA.The expressed protein is then harvested, and purified where desired,using any convenient protocol.

As such, the subject methods provide a means for at least enhancing theamount of a protein of interest in a multi-cellular organism. The term‘at least enhance’ includes situations where the methods are employed toincrease the amount of a protein in a multi-cellular organism where acertain initial amount of protein is present prior to practice of thesubject methods. The term ‘at least enhance’ also includes thosesituations in which the multi-cellular organism includes substantiallynone of the protein prior to practice of the subject methods. As thesubject methods find use in at least enhancing the amount of a proteinpresent in a multi-cellular organism, they find use in a variety ofdifferent applications, including pharmaceutical preparationapplications and therapeutic applications, where the latter is describedin greater detail infra.

Therapeutic applications in which the subject methods find use includegene therapy applications in which the subject methods are used toenhance the level of a therapeutic protein in the host organism andvaccination applications, in which the subject methods are used tovaccinate the host (or develop vaccines for delivery by other methods).As distinct from DNA based expression protocols, the subject RNA basedexpression protocols are uncomplicated by the need for promoter,enhancer, repressor and other regulatory elements commonly associatedwith eukaryotic genes. The subject methods may be used to deliver a widevariety of therapeutic nucleic acids which, upon entry into the targetcell, provide for the requisite enhanced protein level in the host.Therapeutic nucleic acids of interest include nucleic acids that replacedefective genes in the target host cell, such as those responsible forgenetic defect based diseased conditions, by encoding products that aresupposed to be provided to the host by these defective genes; nucleicacids which have therapeutic utility in the treatment of cancer; and thelike. Representative products involved in gene defect disease conditionswhose level may be enhanced by practicing the subject methods include,but are not limited to: factor VIII, factor IX, β-globin, low-densityprotein receptor, adenosine deaminase, purine nucleoside phosphorylase,sphingomyelinase, glucocerebrosidase, cystic fibrosis transmembraneregulator, a-antitrypsin, CD-18, ornithine transcarbamylase,arginosuccinate synthetase, phenylalanine hydroxylase, branched-chainα-ketoacid dehydrogenase, fumarylacetoacetate hydrolase, glucose6-phosphatase, α-L-fucosidase, β-glucuronidase, α-L-iduronidase,galactose 1-phosphate uridyltransferase, and the like. Cancertherapeutic nucleic acids that may be delivered via the subject methodsinclude: nucleic acids that enhance the antitumor activity oflymphocytes by encoding appropriate factors, nucleic acids whoseexpression product enhances the immunogenicity of tumor cells, tumorsuppressor encoding nucleic acids, toxin encoding nucleic acids, suicidefactor encoding nucleic acids, multiple-drug resistance product encodingnucleic acids, ribozymes, DNA ribozymes, DNA/RNA chimeras, interferingRNA and antisense sequences, and the like.

An important feature of the subject methods, as described supra, is thatthe subject methods may be used for in vivo gene therapy applications.By in vivo gene therapy applications is meant that the target cell orcells in which expression of the therapeutic gene is desired are notremoved from the host prior to practice of the subject methods. Incontrast, the naked nucleic acid compositions are administered directlyto the multi-cellular organism and are taken up by the target cells,following which expression of the encoded product occurs.

As mentioned above, another therapeutic application in which the subjectmethods find use is in vaccination of a host (as well as development ofa vaccine to be delivered by other methods). In these methods, the nakednucleic acid, e.g. RNA, that is administered to the host via the subjectmethods encodes a desired immunogen that, upon entry of the RNA into thetarget cell, is expressed and secreted to elicit the desired immuneresponse. Vaccination methods in which naked nucleic acid are employedand in which the subject methods of naked nucleic acid delivery find useare further described in WO 90/11092, the disclosure of which is hereinincorporated by reference.

As mentioned above, the subject methods also find use in variousresearch applications. One research application in which the subjectinvention finds use is in the production of animal models of RNA virusinfection, where RNA viruses of interest include: HCV, HIV, influenza A,Hepatitis A, poliovirus, enteroviruses, rhinoviruses, aphthoviruses, andthe like. To produce such animal models, constructs are first providedthat include one or more regulatory elements from the RNA virus ofinterest operably linked to a reporter domain, e.g., a domain encoding adetectable product (such as luciferase, a fluorescent protein, etc.);etc. Alternatively, DNA constructs that can be transcribed in vivo intosuch RNA constructs may be employed. These constructs are thenadministered to a host, e.g., a mouse, according to the subject methodsto produce an animal model of an infection by the corresponding RNAvirus. As such, also provided are the animal models of RNA virusesproduced by the subject methods. A representative protocol for theproduction of an RNA virus animal model is provided in the experimentalsection infra.

The subject methods also find use in the delivery of RNAi therapeuticand/or research agents, including siRNA and shRNA, as described morefully above and in the experimental section, below.

Also provided are methods of screening candidate modulatory, e.g.,enhancing or inhibitory, agents using such animal models. A variety ofdifferent types of candidate agents may be screened according to thesubject methods. Candidate agents encompass numerous chemical classes,though typically they are organic molecules, preferably small organiccompounds having a molecular weight of more than 50 and less than about2,500 daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Of particular interest in certain embodiments are antisense nucleicacids. The anti-sense reagent may be antisense oligonucleotides (ODN),particularly synthetic ODN having chemical modifications from nativenucleic acids, or nucleic acid constructs that express such anti-sensemolecules as RNA. The antisense sequence is complementary to the mRNA ofthe targeted gene, and inhibits expression of the targeted geneproducts. Antisense molecules inhibit gene expression through variousmechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about16 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra, and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorodiamidatelinkages, methylphosphonates phosphorothioates; phosphorodithioates,where both of the non-bridging oxygens are substituted with sulfur;phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiralphosphate derivatives include 3′-O-5′-S-phosphorothioate,3′-S-5′-O-phosphorothioate, 3′-CH₂-5′-O-phosphonate and3′-NH-5′-O-phosphoroamidate. Peptide nucleic acids replace the entireribose phosphodiester backbone with a peptide linkage. Sugarmodifications are also used to enhance stability and affinity. Oneexample is the substitution of the ribose sugar with a morpholine. Theα-anomer of deoxyribose may be used, where the base is inverted withrespect to the natural β-anomer. The 2′-OH of the ribose sugar may bealtered to form 2′-O-methyl or 2′-O-allyl sugars, which providesresistance to degradation without comprising affinity. Modification ofthe heterocyclic bases must maintain proper base pairing. Some usefulsubstitutions include deoxyuridine for deoxythymidine;5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine fordeoxycytidine. 5- propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

In such screening assays, the nucleic acid construct (e.g., the RNA orDNA construct described above) and the candidate agent are administeredto the host animal, the effect of the candidate agent on the activity ofthe construct is observed, and the observed effect is related to themodulatory activity of the candidate compound. The candidate agent andnucleic acid construct may be administered to the host according to thesubject methods at the same or different times, where in certainpreferred embodiments the two components are administered to the hostsimultaneously, e.g., in the form of a single fluid composition.Representative screening assays are provided in the experimental sectioninfra.

Another research application in which the subject methods find use isthe elucidation of gene function. In such methods, RNA having aparticular gene sequence is introduced via the subject methods and theeffect of the gene on the phenotype of the organism is observed.Benefits of using the subject methods for gene function researchapplications include the ability to express the genes without concernfor genetic regulatory elements. Other research applications in whichthe subject methods find use include, but are not limited to: the studyof ribozyme and antisense efficacy; the study of RNA metabolism, and thelike.

Kits

Also provided by the subject invention are kits for use in practicingthe subject methods of in vivo nucleic acid delivery to a target cell,e.g. hepatic cells. The subject kits generally include a naked nucleicacid that is desired to be introduced into the target cell and an RNaseinhibitor. The subject kits may further include an aqueous deliveryvehicle, e.g. a buffered saline solution, etc. In addition, the kits mayinclude a competitor RNA, as described supra. In the subject kits, theabove components may be combined into a single aqueous composition fordelivery into the host or separate as different or disparatecompositions, e.g. in separate containers. Optionally, the kit mayfurther include a vascular delivery means for delivering the aqueouscomposition to the host, e.g. a syringe etc., where the delivery meansmay or may not be pre-loaded with the aqueous composition. In cases werethe reporter gene is transcribed in vivo from a DNA, RNase inhibitor andcompetitor RNA are not required.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g. a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium, e.g.diskette, CD, etc., on which the information has been recorded. Yetanother means that may be present is a website address which may be usedvia the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental I. RNAi in Mammals

A. We co-delivered a 2 micrograms of plasmid that expresses a luciferasemRNA (pCMVGL3) mixed with 1.8 ml PBS, 1200 units of RNasin and 40micrograms of competitor RNA along with the following formulations:

1) (Group 1 no RNA) 1.8 ml PBS as a untreated control;

2) (Group 2 antisense RNA) 1.8 ml PBS mixed with 20 micrograms ofantisense orientation 21 mer RNA/DNA chimera with the sequence5′-UCGAAGUACUCAGCGUAAGdTdT-3′ (SEQ ID NO:01) (deoxythymidilate residuesare indicated by dT, the remaining nucleotides are ribonucleotides); or

3) (Group 3 RNAi)1.8 ml PBS mixed with 20 micrograms of antisense 21 merdescribed above annealed to 20 micrograms of its sense complement (withsequence 5′-CUUACGCUGAGUACUUCGAdTdT-3′)(SEQ ID NO:02).

The oligonucleotides were kinased using adenosine triphosphate and T4polynucleotide kinase. Each formulation (1-3) was tested by highpressure tail vein injection in 5 week old female Balb/c mice. At 5, 72and 96 hours post injection, light emitted as a result of luciferaseexpression was measured as described above. The results of thisexperiment are summarized in the table below. Numbers expressed asrelative light units.

Group 1 Group 2 Group 3 Group1 standard Group 2 standard Group 3standard no RNA error Antisense error RNAi error  3 hours 1.11 × 10⁹2.05 × 10⁸ 1.29 × 10⁹ 7.90 × 10⁷ 7.90 × 10⁸ 3.54 × 10⁷ 72 hours 6.60 ×10⁶ 7.57 × 10⁵ 5.41 × 10⁶ 9.91 × 10⁵ 8.23 × 10⁵ 2.86 × 10⁵ 96 hours 3.41× 10⁶ 4.50 × 10⁵ 2.72 × 10⁶ 5.25 × 10⁵ 4.61 × 10⁵ 6.77 × 10⁴

The above results demonstrate that RNAi (group 3) caused the destructionof luciferase RNA in the liver of an adult mammal. This destructionresulted in a decrease in light emitted as a result of luciferaseactivity when compared to animals that received no RNA or antisenseoligonucleotide alone. To our knowledge, this is is the firstdemonstration that RNAi is effective in an adult mammal. This methodprovides a model system to study the mechanism by which RNAi functionsin a mammal. It is also useful for the development and optimization ofRNAi based therapeutics. Furthermore, one need not codeliver theexpression plasmid with the modulating agent. One could also deliver amodulating agent targeting an endogenous gene.

B. Here, we test the ability of RNAi to suppress gene expression inadult mammals. We find that synthetic small interfering RNAs (siRNAs)are potent inhibitors of gene expression in vivo. Furthermore,small-hairpin RNAs (shRNAs) are similarly effective. Notably, these RNAiagents can be delivered either as synthetic RNAs or transcribed in vivofrom DNA expression constructs. These studies indicate that RNAi can bedeveloped as a therapeutic tool and demonstrate that it can be employedwith conventional gene-therapy strategies.1. siRNAs

We modified existing hydrodynamic transfection methods J. Chang, L. J.Sigal, A. Lerro, J. Taylor, J Virol 75, 3469-73. (2001)) to permitefficient delivery of naked RNAs. Either an siRNA derived from fireflyluciferase or an unrelated siRNA were co-injected with a luciferaseexpression plasmid (construct description in FIG. 1). Luciferaseexpression was monitored in living animals using quantitative whole bodyimaging following injection of a luciferase substrate (4) and wasdependent on the amount of reporter plasmid injected and the time aftertransfection (data not shown). Representative animals are shown in FIG.2A. Quantification of these results is shown in FIG. 2B.

In each experiment, serum measurements of a co-injected plasmid encodinghuman α-1 antitrypsin (hAAT) (S. R. Yant, et al., Nat Genet 25, 35-41.(2000)) served as an internal control to normalize transfectionefficiency and to monitor non-specific translational inhibition. Averageserum hAAT levels at 74 hours were similar in each group of animals.

Our results indicate specific siRNA-mediated inhibition of luciferaseexpression in adult mice (p<0.0115); unrelated siRNAs were withouteffect (p<0.864). In 11 independent experiments, luciferase siRNAsreduced luciferase expression (emitted light) by an average of 81%(+/−2.2%).

2. shRNA

Short hairpin RNAs (shRNAs) targeting firefly luciferase of renillaluciferase were synthesized by T7 polymerase in vitro runofftranscription. Co-transfection of these in vitro transcribed RNAs withpGL3-Control DNA resulted in reduced firefly luciferase expression inculture (Paddison et al, Genes Dev. 16(8):948-58 (2002)).

In order to test whether these hairpin RNAs were functional in mice, wehydrodynamically transfected 40 μg of in vitro transcribed luciferaseshRNA (or as a control, renilla shRNA), 2 μg pGL3-Control DNA 2 μgpThAAT, 800 units of RNasin and 1.8 ml of PBS into mice. Light emittedfrom mice 72 hours after receiving firefly luciferase shRNAs was reducedby an average of 95% (+/−1.4%) compared to the untreated control. Lightemitted from mice receiving the renilla shRNA was reduced only slightly.Surprisingly, co-transfection of T7 transcription template DNA with aplasmid expressing the T7 polymerase protein did not lead to anyreduction in luciferase reporter activity in culture or in mice (datanot shown).

Firefly Luciferase shRNA sequence (from 5′ to 3′) (SEQ ID NO: 11)GGUCGAAGUACUCAGCGUAAGUGAUGUCCACUUAAGUGGGUGUUGUUUGUG UUGGGUGUUUUGGUURenilla Luciferase shRNA sequence (from 5′ to 3′) (SEQ ID NO: 12)GGGAUGGACGAUGGCCUUGAUCUUGUUUACCGUCACACCCACCACUGGGAGAUACAAGAUCAAGGCCAUCGUCUUCCU

The above results demonstrate that short in vitro transcribed hairpinsalso reduced luciferase expression in vivo.

3. Conclusion

The above data demonstrate that RNAi can downregulate gene expression inadult mice.

C. Hepatitis C virus (HCV) is an RNA virus that infects 1 out of 40people worldwide and is the most common underlying cause for livertransplantation in the western world. To determine whether RNAi could bedirected against a human pathogen, several siRNAs were tested for theirability to target HCV RNAs in mouse liver. We used a reporter strategyin which HCV sequences were fused to luciferase RNA and RNAi wasmonitored by co-transfection in vivo. siRNAs targeting the HCV internalribosome entry site and core protein coding region failed to inhibitluciferase expression. In contrast, siRNAs targeting the NS5B region ofa chimeric HCV NS5B protein-luciferase fusion RNA reduced luciferaseexpression by 75% (+/−6.8%). These results indicate the utility of usingRNAi therapeutically to target important human pathogens.D. From these data, it is clear that siRNAs are functional in mice.Functional shRNAs, which are equally effective in inducing genesuppression, can be expressed in vivo from DNA templates using RNApolymerase III promoters (Paddison et al., submitted). Expression of acognate shRNA (pShh1 -Ff1) induced up to a 98% (+/−0.6%) suppression ofluciferase expression, with an average suppression of 92.8% (+/−3.39%)in three independent experiments (FIGS. 2C and 2D). An emptyshRNA-expression vector had no effect (data not shown). Furthermore,reversing the orientation of the shRNA (pShh1-Ff1rev) insert abolishedsilencing, due to altered termination by RNA polymerase III andconsequent production of an improperly structured shRNA (Paddison etal., submitted). These data indicate that plasmid-encoded shRNAs caninduce a potent and specific RNAi response in adult mice. Furthermore,it demonstrates that this method of RNAi delivery can be tailored totake advantage of the significant progress that has been made in thedevelopment of gene-transfer vectors.

Existing gene therapy strategies depend largely upon the ectopicexpression of exogenous proteins to achieve a therapeutic result. Sinceits discovery, RNAi has held the promise of complementing thesegain-of-function approaches by providing a means for silencingdisease-related genes. Considered together, our results indicate thatRNAi can be induced in adult mammals using DNA constructs to direct theexpression of small hairpin RNAs. These studies demonstrate that thepresent invention provides viral and non-viral delivery systems forapplication of therapeutic RNAi to a wide range of diseases.

II. Hydrodynamic Delivery of Naked RNA A. Introduction

Unless otherwise noted, in all experiments RNAs and DNAs were added tothe indicated amount of RNasin and brought to a final volume of PBSequal to 1.4-1.8 milliliters. This solution was injected into the tailvain of the mice in 4-5 seconds. All RNAs used in these studies weresynthesized using an mMessage Machine kit and purified using an RNeasykit (both from Qiagen Inc.). However, it should not be necessary topurify the RNA and other purification methods exist that should alsowork. RNasin used in all the experiments listed here was native RNasinpurified from human placenta unless otherwise indicated (purchased fromPromega Inc.). For luciferase samples, at the indicated time, mice weregiven an intraperitoneal injection of luciferin (1.5 micrograms/ grambody weight) and the light emitted from the mouse was measured.Background is ˜2×10² relative light units. Human factor IX samples wereanalyzed using an enzyme linked immunoassay.

B. Hydrodynamic Delivery of Naked RNA

RNAs coding for luciferase protein were injected into living mice with:

1) no RNase inhibitor; or

2) RNase inhibitor (called RNasin).

All RNA samples also contained an uncapped unpolyadenylated RNA(competitor RNA) that was included as a competitive inhibitor of RNaseactivity. Total RNA in each sample was adjusted to a total of 80micrograms with competitor RNA. As a negative control (described below)DNAs expressing the luciferase protein under the control of aprokaryotic promoter were also injected. At 3 and 6 hours mice weregiven an intraperitoneal injection of luciferin (the substrate for theluciferase enzyme) and the light emitted from the mouse was measured.

Results summarized in Table 1

TABLE 1 Number Relative Light Nucleic of Mice Units Acid Used (N)Formulation (RLU/5 min) Poly A RNA 1 4 units of 1.0 × 10⁶ RNasin Poly ARNA 1 400 units of 2.0 × 10⁷ RNasin Poly A signal RNA 1 4 units of 7.2 ×10⁴ RNasin Template DNA 1 none signal at background

The above results show that:

-   -   Injected RNA is transfected into the liver of living mice.    -   Capped polyadenylated RNA with a poly A tail (Poly A RNA) is        translated in mouse livers because capped polyadenylated RNA        gives a strong luciferase signal    -   Capped RNA with a poly A signal (Poly A signal RNA) is        translated in mouse livers but it gives a signal but it is about        100 fold lower than that seen with the RNA that has a poly A        tail        The RNAs used in all the experiments described here were        transcribed from a bacterial promoter on a DNA plasmid. This        promoter should not function efficiently in mammalian cells. The        DNA template was removed after transcription using a DNase,        however there is always the concern that the signal seen could        be the result of DNA contamination. To control for this, an        amount of template DNA equivalent to that used in the        transcription was injected. If the signal is due to DNA        contamination then this sample should give a signal. However, no        signal is seen from the DNA control.

It was also found that addition of an RNase inhibitor (called RNasin)protects the RNA from degradation by serum nucleases, thus increasingthe observed signal, because addition of RNasin increased the signal by20 fold at the dose used.

From the above, the following conclusions are drawn. Hydrodynamicdelivery of naked RNA results in high level transfer of RNA into thelivers of living mice. Furthermore, capped and polyadenylated RNA worksbetter than RNA with a polyadenylation signal but no poly A tail,although both RNAs gave a signal. Addition of an RNase inhibitorprotected the RNA from degradation, resulting in a higher luciferasesignal. Finally, the signal seen with the injected RNA is not due to DNAcontamination.

C. Refinement of System

RNAs coding for luciferase protein were injected into living micewith 1) high or low doses of native or recombinant RNasin or 2) aftertreatment with RNase T1 which should destroy the RNA and abolish thesignal (negative control). All RNA samples also contained an uncappedunpolyadenylated competitor RNA such that the total amount of RNAinjected was 80 micrograms. Control DNAs expressing the luciferaseprotein under the control of a prokaryotic promoter were also injectedin indicated control reactions. At 3 and 6 hours mice were given anintraperitoneal injection of luciferin and the light emitted from themouse was measured. This experiment is largely to verify the results ofthe first experiment and to test which parameters are important. At thesix hour timepoint, one mouse that had been injected with RNA wassacrificed and its organs were removed to determine which organs expressluciferase.

The results are summarized in Table 2

TABLE 2 micro- Relative Light Relative Light Relative Light grams NumberUnits Units Units Nucleic Acid of RNA of Mice (RLU/5 min) (RLU/5 min)(RLU/5 min) Used or DNA (N) Formulation 3 hours 6 hours 24 hours Poly ARNA 35 1 240 units 1.8 × 10⁵ 1.1 × 10⁶ Background RNasin (Native) Poly ARNA 50 1 240 units 1.6 × 10⁶ 5.4 × 10⁵ Background RNasin (Native) Poly ARNA 50 1 44 units 5.5 × 10⁴ 1.9 × 10⁴ RNasin (Native) Poly A RNA 10 1240 units 7.7 × 10⁴ 1.8 × 10⁵ RNasin (Recombinant) Poly A RNA 50 2 3000units Background Background RNase T1 Template 2 1 none BackgroundBackground DNAThe above results demonstrate that:

-   -   The dose of RNasin alters the level of expression seen because        increasing doses of RNasin lead to increased levels of        luciferase activity.    -   Both native and recombinant RNasin both protect the RNA.    -   When the RNA is destroyed with RNase, the signal is abolished,        demonstrating that the RNA is responsible for the signal        (negative control).    -   When an amount of template DNA equivalent to that used in the        transcription is injected without DNase treatment, no signal is        seen, demonstrating that the signal is not due to DNA        contamination.    -   Liver is the only site of luciferase expression.

From the above, the following conclusions are drawn. RNasin dose effectsthe level of expression. Both recombinant and native RNasin protect theinjected RNA. No signal was seen when template DNA was injected or whenRNA was destroyed with RNase, demonstrating that signal is not theresult of DNA contamination. Finally, liver is the only site ofluciferase expression.

D. Competitor RNA Enhances the Activity.

Luciferase activity from 20 micrograms of capped and polyadenylatedluciferase RNA was measured. Four conditions were tested in experimentssimilar to those described in experiments 1 and 2:

1) 400 units of RNasin +competitor RNA;

2) 40 units of RNasin with no competitor RNA;

3) 800 units of RNasin with no competitor RNA;

4) 1200 units of RNasin with no competitor RNA.

At 3, 6 and 9 hours mice were given an intraperitoneal injection ofluciferin and the light emitted from the mouse was measured.The results are summarized in Table 3.

TABLE 3 Micro-grams Number Average Average Average Competitor Units ofof Mice (RLU/2 min) (RLU/2 min) (RLU/2 min) RNA RNasin (N) 3 hours 6hours 9 hours RLU 60 400 3 7.6 × 10⁴ 1.7 × 10⁴ 3.5 × 10³ standard 3.5 ×10⁴ 4.2 × 10³ 9.6 × 10³ error RLU None 400 3 6.5 × 10³ 4.2 × 10³ 2.6 ×10³ standard 1.4 × 10³ 2.8 × 10³ 1.7 × 10³ error RLU None 800 3 6.2 ×10⁴ 8.7 × 10³ 2.0 × 10³ standard 3.1 × 10⁴ 2.5 × 10³ 3.7 × 10³ error RLUNone 1200 3 7.6 × 10⁴ 2.2 × 10⁴ 7.4 × 10³ standard 5.4 × 10⁴ 1.6 × 10⁴4.5 × 10³ errorThe above results demonstrate that:

-   -   RNasin dose alters the luciferase activity because increasing        doses of RNasin lead to increasing luciferase activity. The        highest dose (1200 units of RNasin) gave the highest activity at        all times tested.    -   The addition of competitor RNA enhanced the measured luciferase        activity, because presence of the competitor RNA enhanced the        luciferase activity. This effect was synergistic with the        protective effect of the RNasin.

From the above results, the following conclusions are drawn. Addition ofcompetitor RNA increases luciferase signal. Furthermore, increasingdoses of RNasin lead to increasing levels of luciferase activity

E. Cap Independent Translation of Luciferase using an Internal RibosomeEntry Site.

In Eukaryotes, translation of RNAs into protein occurs by two differentmechanisms called cap dependent and cap independent translation. Capindependent translation requires a 5′ nontranslated region called aninternal ribosome entry site (IRES). Several RNA viruses, such ashepatitis C virus (HCV), polio virus and hepatitis A utilize IRESsequences to carry out cap independent translation. We originallydeveloped the RNA transfection method described here with the idea thatit could be used to make a small animal model system for studyinganti-HCV therapeutics. Transfection with IRES RNAs could also be usedfor mutagenesis studies designed to investigate sequence elementsnecessary for efficient IRES function.

1. Description of Experiment and Results:

The RNA HCVluc has the HCV IRES at the 5′ end and the luciferase genefollowed by a poly A tail. 40 micrograms of HCVluc+40 micrograms ofcompetitor RNA+20 microliters of RNasin were injected into the tail vainof the mice. At 3 and 6 hours mice were given an intraperitonealinjection of luciferin and the light emitted from the mouse wasmeasured. Result: The HCV IRES was able to drive translation of theinjected HCV luciferase RNA fusion. Quantitation of the results issummarized in Table 4.

TABLE 4 3 hours 6 hours post post injection injection Average RelativeLight Units 1.7 × 10⁵ 4.6 × 10⁴ Standard Error 7.4 × 10⁴ 1.6 × 10⁴F. Measurable Serum Concentrations of Human Factor IX (hFIX) Protein canbe Produced and Secreted Upon Injection of hFIX RNA.

Human factor IX protein is a blood clotting protein that is not producedby some patients with hemophilia. The levels of this protein in serumcan be easily measured using an enzyme linked immunoassay (ELISA). Wechose to express this protein for two reasons:

1) hFIX is a therapeutically relevant protein. Although transientexpression of hFIX is not clinically relevant, it would be desirable totransiently express some other types of therapeutic proteins that do notrequire chronic expression.2) hFIX is a human protein and is thus capable of eliciting an immuneresponse in mice.

One application of RNA injection is in the development and testing ofvaccines. An immune response to hFIX upon injection of hFIX RNA woulddemonstrate the proof of principle of using RNA as a vaccine.

1. Description of Experiment and Results:

40 micrograms of capped and polyadenylated hFIX RNA+40 micrograms ofcompetitor RNA+800 units of RNasin were injected by tail vain into 1mouse. Result: 40 nanograms/milliliter of serum were detected by ELISAat 6 hours. This amount of hFIX is within the significant range of theELISA assay.

G. Hydrodynamic Delivery of HCV Genomic RNAs to Create an HCV MouseModel

Two groups of 6 mice were injected with:

1) 50 micrograms of capped HCV full length genomic RNA called 90 FL HCV(which also contains some uncapped RNA)+40 micrograms of capped andpolyadenylated hFIX RNA+400 units of RNasin; or

2) a full length non-infectious HCV genomic RNA that has a mutation inthe replicase gene that makes it catalytically inactive (called 101 FLHCV)+40 micrograms of capped and polyadenylated hFIX RNA+400 units ofRNasin.

The transcription templates for making the HCV RNAs were obtained fromCharles Rice and Washington University. Six hours after injection themice were bled and hFIX levels are being measured to normalize forinjection efficiency. The injected HCV RNAs are expected to degraderapidly. Any RNA detected after a few days is likely to be RNA newlysynthesized during viral replication. A quantitative real time PCRmethod has been developed to measure the levels of HCV RNA in the liversof these mice. If replication of the virus occurs, then the levels ofHCV RNAs in the mice injected with 90 FL HCV will be greater than thelevels in mice injected with 101 FL HCV when measured weeks afterinjection. A histological assay is also being developed in order toassay for the synthesis of HCV proteins. Three different positiveoutcomes are possible 1) The RNA enters the liver but is not translatedand does not replicate 2) the RNA enters the liver and is translated butdoes not replicate 3) the RNA enters the liver, is translated andreplicates. All three outcomes are useful model systems. If 1, 2 or 3occurs then this system could be used to test ribozymes directed againstHCV RNAs (see experiment 9 below). If 2 or 3 occurs then, the thissystem could be used to test inhibitors of HCV translation, replicationand infection.

Injection of this RNA did not result in a viral replication cycle forHCV. However, another group has used a similar method to initiate ahepatitis delta replication cycle. See Chang J, Sigal LJ, Lerro A,Taylor J., J Virol.75(7):3469-73 (2001).

H. In Vivo Cleavage of HCV RNAs by Ribozymes

DNAzymes targeting the IRES of HCV have been chemically synthesized. Wehydrodynamically injected these ribozymes into mice and assessed theirability to decrease the levels of injected HCV RNAs within the liver.Five nanomoles of DNAzyme targetting the IRES was coinjected with 20 μgof an RNA comprised of the HCV IRES followed by the firefly luciferasecoding sequence followed by 30 adenosines. The sequence of the DNAzymewas 5′-GAGGTTTAGGAGGCTAGCTACAACGATCGTGCTCA-3′ (SEQ ID NO:013). Mice thatreceived the DNAzyme in combination with the target RNA emitted 95% lesslight at 6 hours than mice that received the target RNA alone.Conclusion: We demonstrated that this DNAzyme can inhibit translationfrom the HCV IRES, presumably by cleaving the IRES RNA sequence.Synthetic ribozymes were also tested using an analogous methodology andwere found to be ineffective.I. This experiment is to do a timecourse of luciferase expression aftera single injection of capped and polyadenylated RNA. If the followingcondition is met, then we can use a first order exponential decay fit(described by Equation 1) of the data to calculate the degradation rateof the expressed protein. In order for this data to be fit to a simplefirst order exponential decay, the half life of the mRNA must besignificantly less than the halflife of the protein (at least 5-10 foldless). If this condition is not met, then a more complex mathematicalrelationship that takes into account the halflife of the mRNA can beused. Another solution to this problem is to decrease the half life ofthe mRNA by making it uncapped or omiting the competitor RNA.

If we define the amount of protein at a given time (or the signal fromthe protein) as A, the amount of protein (or signal) at the firsttimepoint as Ao, the decay rate constant as k and time after the firstmeasurement as t, the equation would be of the form:

A=Ao exp^((−kt))   (Equation 1)

1. Description of the Experiment:

Four groups of 6 mice were injected with 20 micrograms of cappedpolyadenylated luciferase RNA+60 micrograms of uncapped competitorRNA+800 units of RNasin. At 3, 6, 9 or 24 hours, the mice were given anintraperitoneal injection of luciferin (1.5 micrograms/gram body weight)and the light emitted from the mouse was measured.

The results are provided in the table below:

Hours Post Light Units Standard Standard Error 1 3.000 530000.000330000.000 150000.000 2 6.000 200000.000 88000.000 36000.000 3 9.000110000.000 43000.000 18000.000 4 24.000 1900.000 1100.000 440.000Relative light units were plotted vs. time and the resulting curve isfit to Equation 1. This analysis yields an apparent degradation rateconsant of 0.297 hour⁻¹.

The most common method for measuring a half-life of a protein is thefollowing. In one approach, the protein is purified and sometimeslabeled (for example with radioactive iodine). The purified protein isinjected and at different times the animal is sampled and the amount ofprotein remaining at any given time is plotted vs. time and the curve isfit to an equation such as Equation 1. The advantage of our method isthat it does not require the in vitro synthesis or purification of theprotein.

J. We have constructed RNAs that contain regulatory regions of the HCVRNA controlling the translation of a protein called luciferase (referredto here as HCV luc RNA). We have also constructed DNA expressionplasmids that express similar RNAs once they enter cells (referred tohere as HCV luc DNA). See FIG. 3 for diagrams of these constructs.

When either the HCV luc RNAs or the HCV luc DNAs are transfected intomice, they go to the liver and HCV luc RNAs or RNAs transcribed from theHCV luc DNAs are translated into luciferase protein. At various times,the substrate of the luciferase protein, luciferin, is injected into themice. The enzyme luciferase consumes the luciferin and makes light inthe process. The amount of light emitted from the mice is proportionalto the amount of luciferase protein present at the time of the sampling.

We have synthesized short synthetic oligonucleotides of a type known asMorpholino oligos. We mixed 1 nanomol of a morpholino oligo with 10micrograms of HCV luc RNA or 1 microgram of HCV luc DNA. The morpholinooligo was made by Gene Tools, LLC in Corvallis, Oregon and has thesequence 5′-TCTTTGAGGTTTAGGATTCGTGCTC-3′ (SEQ ID NO:14). This mixture isthen added to 1.8 milliliters of buffer and injected under high pressureinto the tail veins of mice as described in our previous application. Asa control, mixtures that do not contain the inhibitor are injected intoother mice. In the presence of inhibitor, emitted light is reduced bymore than 90%. We conclude from this finding that translation of theinjected RNA or translation of the RNA produced from the injected DNA isprevented by the inhibitor by an antisense mechanism. In the case of theinjected RNA we can only follow this inhibition for about 24 hours,because of the limited stability of the RNA in cells. In the case of theinjected DNA, we can monitor translation for about 8 days. Thetranslational inhibition lasted for the whole duration of the time wecould measure translation in this system.

K. Experiment A

control group: RNAs containing the HCV IRES and a luciferase reportersequence are injected into mice and they glow when this RNA istranslated into luciferase proteinTest group:Coinject inhibitor with RNA. Both go to the same cells. Inhibition isexpressed as activity (glowing) compared to control group.

Experiment B

Same as experiment A except we inject a DNA that encodes the target RNAalong with the inhibitor. The DNA goes to the nucleus of the mousehepatocytes and is transcribed to give the target RNA. This RNA goes tothe cytoplasm of the cells where it interacts with the inhibitor.

The constructs employed in these experiments are provided in FIG. 4. Theresults of these experiments with antisense and DNAzyme inhibitors areprovided in FIGS. 5A to 5F.

The above screening protocol in which the inhibitor and RNA/DNA arecoadministered offers important advantages in terms of allowing one toseparate issues of drug delivery from issues of drug efficacy.

It is evident from the above results and discussion that the subjectinvention provides a viable way of using RNAi agents in non-embryonicmammalian organisms, where the subject methods and compositions can beemployed for a variety of different academic and therapeuticapplications. In addition, the subject invention provides an improvedmethod of transferring a nucleic acid into a target cell is provided bythe subject invention. Specifically, the subject invention provides fora highly efficient in vivo method for naked nucleic acid transfer whichdoes not employ viral vectors and therefore provides many advantagesover prior art methods of nucleic acid transfer. As such, the subjectinvention represents a significant contribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method of reducing expression of a target RNA in a target cell of anon-embryonic mammal in vivo, said method comprising: administering tosaid mammal an effective amount of an siRNA that is specific for saidtarget RNA to reduce expression of said target RNA in said target cell.2-7. (canceled)
 8. The method according to claim 1, wherein saidnon-embryonic mammal is an adult.
 9. The method according to claim 1,wherein said non-embryonic mammal is a juvenile. 10-20. (canceled) 21.The method according to claim 1, wherein said target cell is a hepaticcell.
 22. The method according to claim 1, wherein said method furthercomprises administering an RNAse inhibitor. 23-37. (canceled)
 38. Themethod according to claim 1, wherein the target RNA is an endogenousmRNA.
 39. The method according to claim 1, wherein the target RNA ispathogen RNA.
 40. The method according to claim 39, wherein the pathogenis a virus.
 40. The method according to claim 1, wherein the siRNA ischemically modified.
 41. The method according to claim 40, wherein thechemically modified siRNA comprises one or more backbone modificationsselected from the group consisting of: a phosphorodiamidate, amethylphosphonate, a phosphorothioate, a phosphorodithioate, aphosphoroamidite, an alkyl phosphotriester, and a boranophosphate. 42.The method according to claim 41, wherein the chemically modified siRNAcomprises a sugar modification.
 43. The method according to claim 42,wherein the chemically modified siRNA comprises a 2′-O-methyl sugarmodification.